A significant challenge for the development of new influenza vaccines is to identify strategies that can both accelerate vaccine production and protect against the emergence of epidemic and pandemic strains. This proposal will develop a DNA vaccine to meet these needs. DNA vaccines can be rapidly designed and manufactured to express multiple antigens and induce antibody and cell-mediated protection against distant drift variants. This proposal will employ particle-mediated epidermal delivery (PMED) of the DNA vaccine and builds on the recent success of a seasonal influenza PMED DNA vaccine that induced protective levels of antibody in vaccinated subjects in a phase I human clinical trial. The purpose of this proposal is to increase PMED DNA vaccine immunogenicity and further develop the vaccine as a pandemic flu product. The primary objectives of the proposal are to: 1) Broaden the specificity of the vaccine against genetically drifted strains and avian influenza by including multiple HA sequences. 2) Investigate the effects of including a nucleoprotein (NP) gene in the multivalent DNA vaccine with sequences modified and optimized to increase the breadth and potency of CD8+ T cell responses against highly conserved regions of the gene. 3) Determine if a novel genetic adjuvant that we have shown enhances antibody and cellular immune responses against other DNA vaccine antigens will similarly increase the immunogenicity and protective efficacy of the candidate pandemic influenza DNA vaccine. The vaccine will be tested for immunogenicity in the highly relevant ferret and nonhuman primate preclinical models. A model in rhesus macaques will be developed to take advantage of the information available on the Class I locus of this species required for analysis of T cell immunity. Aerosolized influenza challenges will be employed so that protective efficacy against homologous and drifted strains of avian influenza can be evaluated in a setting that mimics natural exposure in the population. These efforts will generate three candidate DNA vaccine products that induce antibody and cellular immune responses for protection against both seasonal and pandemic strains of influenza. These experiments will also test the hypothesis that vaccine induction of CD8+ T cell responses against conserved regions of the virus will increase protective efficacy against HA-drift variants and provide critical protection against mortality caused by emerging pandemic strains of influenza viruses.